Although mitochondrial gene expression depends on nuclear genome function and reciprocally "retrograde communication" from mitochondria regulates nuclear gene expression, the mechanisms coordinating these events remain to be clarified. Mitochondria are targets of estrogen action. However, the mechanisms for estrogenic effects on mitochondrial function are not yet defined. The overall goal of this project is to determine the mechanism(s) by which estradiol (E2) and selective estrogen receptor (ER) modulators (SERMs) regulate the transcription of Nuclear Respiratory Factor-1 (NRF-1) and the downstream impact of NRF-1 on estrogen- and SERM- regulated mitochondrial activity in normal and neoplastic cells. NRF-1 is a transcription factor that is critical for integrating nucleo-mitochondrial function by initiating transcription of nuclear-encoded mitochondrial DNA (mtDNA) -specific transcription factors including Tfam (mtDNA maintenance factor). We recently reported that E2 induces NRF-1 transcription in MCF-7 breast cancer cells via ER1-estrogen response element (ERE) activation. In Aim 1 we will test the hypothesis that the direct interaction of ER1 and ER2 with the NRF-1 promoter recruits different coregulators in a ligand- and cell- specific manner. Chromatin immunoprecipitation (ChIP) assays will address this hypothesis. The effect of siNRF-1 on E2-regulated cell cycle and apoptotic proteins and cell cycle entry will be examined. Results will reveal if E2-induced NRF-1 plays a role in E2's anti-apoptotic activity and if there are cell-specific differences in these responses. Additionally, we will examine NRF-1 protein expression by IHC in normal and neoplastic human breast specimens. Experiments in Aim 2 will determine the effect of E2 and SERMs on NRF-1 and downstream target gene expression and ER-NRF-1 promoter interaction in vivo. ChIP assays will determine if E2 increases the direct interaction of ER1, coactivators, and RNA pol II with the NRF-1 promoter in vivo in MCF-7 tumor xenografts and mouse tissues, focusing first on uterus. Aim 3 is the `functional outcome'aim to measure the genes/proteins/mitochondrial activity, including O2 consumption, mt biogenesis, heat generation, i.e., downstream effects of E2-induced NRF-1 expression. Experiments will test the hypothesis that the E2- stimulated increase in NRF-1 will, in turn, increase the expression of NRF-1 regulated genes, e.g., TFAM, that subsequently increase expression of its mtDNA-encoded target genes, e.g., Cytochrome c oxidase subunit I (MTCO1) and anti-apoptotic genes, e.g., survivn. Together the results will define the molecular mechanisms of estrogen action coordinating mitochondrial function. Understanding how E2 and SERMs regulate mitochondrial function could lead to new insights in the prevention and progression of breast and lung cancer. PUBLIC HEALTH RELEVANCE: Although mitochondrial gene expression depends on nuclear genome function and reciprocally by "retrograde communication" mitochondrial activity regulates nuclear gene expression, the mechanisms coordinating these events remain to be clarified. Mitochondrial dysfunction and increased reactive oxygen species mediate the pathophysiologic mechanisms of aging, acute neurodegeneration caused by trauma, stroke, or cardiac arrest;and cancer initiation and tumor progression. The impact of steroids on mitochondrial function "is a new and novel area of investigation" and the proposed study seeks to delineate a new biological pathway of estrogen and antiestrogens/SERMs in regulating nuclear respiratory factor-1 (NRF-1) and its downstream targets in mitochondrial activity.